Bacterial colonization, which in many gram-negative bacteria is mediated by extracellular fibers termed pili, is the initial step in many infectious diseases. There are many important types of pili including adhesive pili assembled by a molecular machine called the chaperone/usher system. A single bacterial genome can encode as many as twelve such systems. Pili carry out unique functions that allow pathogenic gram negative bacteria to bind, invade, colonize and form biofilms within host tissues. For example, pili are critical in the ability of uropathogenic E. coli to cause urinary tract infections (UTIs). UTIs are among the most common bacterial infections and nearly 50% of women will be afflicted by at least one UTI in their lifetime with many experiencing recurrent UTIs. We have discovered that the incomplete immunoglobulin (Ig) fold of pilus subunits is transiently completed by the chaperone in a process that catalyzes folding that we termed donor strand complementation (DSC). The outer membrane usher then catalyzes the exchange of the chaperone for subunit-subunit interactions in a process that we termed donor strand exchange (DSE). During DSE, an N terminal extension (Nte) of a subunit completes the Ig fold of an interacting subunit partner in the polymerizing fiber. Using X-ray crystallography and mutagenesis we have visualized and verified the details of chaperone/subunit and subunit-subunit interactions in DSC and DSE, respectively. Based on our recently solved crystal structure and topology mapping, the usher is comprised of four critical domains: the N terminal and C terminal periplasmic domains, the 2 barrel transmembrane channel, and the plug domain that gates the channel. We propose to elucidate the function of each of these usher domains and the mechanisms of usher catalyzed DSE and channel gating. The usher binds discriminately to chaperone/subunit complexes, binding with highest affinity to the chaperone/adhesin complex. This high affinity interaction works in concert with an interaction of the usher with an initiator chaperone/subunit complex, in order to activate the usher into a conformation that allows it to catalyze DSE reactions. We will make domain deletion and single domain variants of the PapC usher, express and purify these PapC variants and use in vitro and in vivo binding assays, including overlay, ELISA, co-purification and interactions measured by fluorescence changes, to assess the ability of the various usher domains to discriminately bind chaperone/subunit complexes. Based on PapC135-640, PapG1-196, PapDGpilin-domain, PapDF, PapDE, PapDK, PapDA, PapDH, and FimCH crystal structures, we will target residues for mutation that appear to be involved in chaperone/subunit/usher interactions and investigate the molecular rules governing the gating of the usher pore, DSE reactions, and usher activation. Also, we will use a library of small molecules called pilicides to probe critical interactions in chaperone/usher assisted pilus assembly. This work will provide the basis for identifying and developing new therapeutic strategies for combating common gram-negative bacterial diseases such as urinary tract infection. PUBLIC HEALTH RELEVANCE: Bacterial fibers called pili, assembled by the chaperone/usher pathway, frequently mediate critical interactions that allow pathogenic gram negative bacteria to bind, invade, colonize and form biofilms within host tissues. Understanding the molecular basis of pilus biogenesis will continue to provide dramatic insights into how pathogenic bacteria produce, secrete and assemble virulence factors. This will lead to the identification and development of small molecular weight inhibitors of these critical pathogenic processes, which hold promise in facilitating the design of new therapeutics for treating many common bacterial infections such as urinary tract infections.